Lipoproteins are aggregates of lipids and protein which circulate in the blood and are the means by which lipids are transported within the body. The lipid portions of these aggregates consist essentially of cholesterol and triglyceride. Serum lipoproteins are classified according to their density. These classes include very low density lipoproteins (VLDL), also known as pre-beta lipoproteins; low density lipoproteins (LDL), also known as beta-lipoproteins; and high density lipoproteins (HDL), also known as alpha-lipoproteins. A fourth class of lipoproteins is chylomicron (CHYLO), stable droplets containing 86% triglyceride fat, 3% cholesterol, 9% phospholipids, and 2% protein. Chylomicrons are found in the intestinal lymphatics and blood during and after meals, and are the form in which absorbed long-chain fats and cholesterol are transported from the intestine.
One of the functions of lipoproteins is to carry water insoluble substances, such as cholesterol and cholesterol esters, for eventual cellular utilization. While all cells require cholesterol for growth, excess accumulation of cholesterol by cells is known to lead to certain diseases, including atherosclerosis. It is now known that the amount of total serum cholesterol can be correlated with the incidence of atherosclerosis. However, since all lipoprotein classes contain varying amounts of cholesterol, total serum cholesterol determination is a complex average of the amount that each lipoprotein class contributes to the total lipoprotein population of the serum.
Recent studies have implicated LDL as the class of lipoproteins responsible for the accumulation of cholesterol in cells, whereas HDL has been shown to be important in the removal of excess cholesterol from cells. Additionally, the correlation of atherosclerosis and the levels of LDL cholesterol is much higher than a similar correlation between atherosclerosis and total serum cholesterol levels. Conversely, there seems to be a negative correlation of atherosclerosis and HDL cholesterol levels. See Goffman, J. W. et al , Circulation, 2: 161-178 (1950); Barr, D. P. et al., Am. J. Med., 11: 480-493 (1951); Nikkala, E., Scand. J. Clin. Lab. Invest. Supplement, 5: 1-101 (1952); Jencks, W. P. et al., J. Clin. Invest., 35: 980-990 (1956), and Miller, G. J. et al., Lancet, 1(7897): 16-19 (1975).
Thus, because the various classes of lipoproteins contain cholesterol and triglyceride in different proportions, determination of only total cholesterol and total triglyceride is not sufficient to differentiate abnormal lipoprotein patterns. Recognition of this fact has led investigators to various procedures designed to determine concentrations of specific lipoproteins rather than just lipids. U.S. Pat. No. 4,126,416 to Sears describes a method for determining the level of LDL cholesterol in blood plasma, the LDL cholesterol being separated from other soluble cholesterol fractions by selectively agglutinating LDL with a plant lectin, followed by detection of the amount of cholesterol associated with the agglutinated LDL.
U.S. Pat. No. 4,167,467 to Golias describes an electrophoresis method for determining the concentration of HDL free cholesterols in body fluids and simultaneously determining the concentration of VLDL and LDL free cholesterols in the fluid sample. The method includes applying a direct current across the fluid medium, applying a developing substrate to the electrophoresed lipoproteins, and quantitatively determining the concentration of each lipoprotein free cholesterol. The method of Golias purports to be an improvement over the prior art in that direct and simultaneous measurement of each lipoprotein free cholesterol fraction is achieved without precipitation of each fraction.
U.S. Pat. No. 4,185,963 to Heuck describes a method for determining lipids in blood serum wherein the VLDL, CHYLO, and HDL are extracted from the serum with a polycation, followed by measuring the lipid content of the LDL in the serum.
U.S. Pat. No. 4,215,993 to Sanders describes a method for isolating HDL from LDL in human serum, followed by quantitative determination of HDL cholesterol. LDLs are precipitated from the serum without the addition of metal ions to the sample. The precipitating reagent lowers the pH of the human serum approximately to the isoelectric point of the LDL through the use of an organic buffer.
U.S. Pat. No. 4,309,188 to Bentzen describes a separation method wherein LDL and HDL are separated on a microcolumn containing a support which has a sulphated polysaccharide covalently bound thereto. Elution with a first pH buffered solution collects the LDL; elution with a second pH buffered solution collects the HDL. Subsequently, LDL/HDL ratios can be determined.
U.S. Pat. No. 4,039,285 to Teipel discloses a single-sample method for determining concentrations of individual lipoprotein classes and lipids in blood by turbidimetric measurement. The ionic strength of the mixture is raised in steps to cause progressive dissolution of each class of complex from that of the highest density lipoprotein to the lowest density lipoprotein. Measurement of the turbidity due to the insoluble complexes present at each step allows the concentration of each lipoprotein class and lipid in the blood sample to be calculated.
Recent epidemiological studies on cardiovascular illness have shown the advantage of determining not only the global amount of serum lipoproteins and distinction according to the group to which these lipoproteins belong, but also, within these groups, according to the type of apolipoprotein (ALP) present, and especially the amount of each ALP present. Apolipoprotein is the protein moiety which binds the lipid moiety to form the holoprotein. At present, a number of types and subtypes of ALP have been identified.
Apolipoprotein A (Apo-A) includes subtypes A.sub.1 and A.sub.2. Apo-A.sub.1 is the major apolipoprotein of HDL and is thought to occupy a surface position on HDL particles, surrounding a neutral lipid core. It is also known that Apo-A.sub.1 activates lecithin:cholesterol acyl transferase, the cholesterol-esterifying enzyme of plasma involved in the production of mature circulating HDL. As mentioned above, there is an inverse correlation between plasma HDL levels and development of coronary artery heart disease. See also, Heiss, G. et al., Circulation, 62:Suppl. IV, 116 (1980).
The second most abundant apolipoprotein of HDL is Apo-A.sub.2. It has been reported that Apo-A.sub.1 binds less total HDL lipid than does Apo-A.sub.2 ; however, in an interaction between these apolipoproteins, Apo-A.sub.2 increases the binding capacity of Apo-A.sub.1. Morrisett et al., "Lipoproteins: Structure and Function," Annual Review of Biochemistry, 44: 183, 196-198 (1975).
Highly purified LDL has been shown to contain a single molecule of a very large protein, apolipoprotein B (Apo-B), having a molecular weight estimated to be 250,000 to 500,000 daltons. See Smith, et al., J. Biol. Chem., 247: 3376 (1984) and Milne, R. W. and Marcel, I. L., FEBS Lett., 146: 97 (1982). LDL plays a key role in the transport of cholesterol to the peripheral tissues where it is bound to cellular receptors and ingested by an endocytosis process. LDL is also known to play an important role in the pathological uptake and deposition of cholesterol, with very high concentrations of LDL implicated as the causative agent of some forms of human atherosclerosis. Additionally, moderate elevations of LDL over long periods of time may be an important factor in the development of most human atherosclerosis. See Goffman et al., Science, 111: 166 (1951); Goldstein et al., Metabolism, 26: 1257 (1977). Apo-B is known to play a number of important roles in triglyceride and cholesterol transport and is required for the formation and secretion of triglyceride-rich lipoproteins from human liver. It is the only protein always found on LDL and contains a site complementary to, and recognized by, the LDL receptor. There is also evidence demonstrating that the presence of a certain allele of pig Apo-B correlates strongly with lipid deposition and plaque formation in pig artery. See Rapacz et al., Exp. and Mol. Path., 27: 429 (1977).
Apolipoprotein C (Apo-C) includes subtypes Apo-Cl, Apo-C.sub.2, and Apo-C.sub.3. Apo-C has been shown to be part of the protein moiety of plasma lipoproteins (Eisenberg, S. et al., J. Biol. Chem., 254: 12603 (1979)). Apo-C, which makes up 40-80% of the total protein of CHYLO and VLDL, is present in plasma HDL, and plays an important role in the regulation of the activity of the enzyme system lipoprotein lipase. Recently, a relationship between the extent of VLDL triglyceride hydrolysis and the content of Apo-C in the lipoprotein has been established, with Apo-C molecules being transferred from VLDL to HDL following abrupt triglyceride hydrolysis, and returning to VLDL when newly secreted particles enter the circulation. Similar observations have been reported during clearance and induction of alimentary chylomicronemia.
One apolipoprotein particularly central to the removal or uptake process for circulating cholesterol-laden lipoproteins is apolipoprotein E (Apo-E). See Mahley, R. W., Med. Clin. North. Amer., 66: 375 (1982). An important function of Apo-E is its mediation of cellular uptake of lipoproteins through specific surface receptors. See Mahley, R. W., Klin. Wochenscher., 61: 225 (1983). Apo-E is known to bind to the low density lipoprotein receptor of fibroblast and various peripheral cells, thereby affecting intracellular cholesterol metabolism. It also binds specifically to a hepatic plasma membrane receptor, the Apo-E receptor, and functions as a prime determinant in chylomicron remnant clearance.
Apolipoprotein E (Apo-E) includes three major iso forms, Apo-E.sub.2, Apo-E.sub.3, and Apo-E.sub.4. Amino acid sequence analysis has demonstrated that the three iso forms differ in their primary structure. Variant forms of Apo-E.sub.2 have been described, with all forms of Apo-E.sub.2 demonstrating reduced LDL receptor binding activity and reduced Apo-E receptor binding activity. Further, these abnormal forms of Apo-E.sub.2 are associated with the genetic abnormality type III hyperlipoproteinemia, which appears to be partly due to the defective clearance of cholesterol-rich remnant lipoproteins (Weisgraber, H. K. et al., J. Biol. Chem., 258: 12341 (1983)). This evidence suggests that Apo-E performs a critical role in cholesterol and lipid metabolism as well.
Based on the recently recognized evidence relating to the interaction of various cellular receptors and ALPs in mediating removal of cholesterol-containing lipoproteins from circulation, efforts have mounted to develop specific assays for ALPs. U.S. Pat. No. 4,399,217 to Holmquist et al. describes a process for the determination of serum lipoproteins by an immunoenzymatic method. Apolipoprotein antibodies are fixed on a support. Serum sample is added, in combination with enzyme-labeled specific apolipoprotein. Elimination of all reagent not fixed on the support, followed by measurement of the enzymatic activity bound to the support, produces an indirect determination of the amount of specific apolipoproteins present in the sample being analyzed in a competitive assay. Thus, the assay requires "type-specific" antibody and specific labeled antigen (apolipoprotein) and a competitive assay system. The "type-specific" antibody is produced by immunizing rabbits with purified apolipoprotein obtained by serum lipoprotein fractions separated by ultracentrifugation on a density gradient. Unfortunately, ultracentrifugation is somewhat deficient with regard to obtaining highly pure apolipoprotein fractions. Accordingly, the "type-specificity" of the resulting antibodies produced by rabbit immunization is deficient as well. Thus, a need has continued to exist for a highly accurate, truly type-specific assay for apolipoproteins and high specificity antibodies for the same.